Reduced cellular levels of DNA polymerase delta alter replication-fork dynamics and enzymology, and globally impair lagging-strand processing

DNA polymerase delta (Pol ?) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol ? is responsible for the synthesis and processing of the lagging strand; this role requires Pol ? to extend Okazaki fragment primers synthesized by Pol ?-primase, and to carry out strand-displacement synthesis coupled to nuclease cleavage during Okazaki fragment termination. Destabilizing mutations in human Pol ? subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol ? in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze the how the depletion of Pol ? impacts replication initiation and elongation in vivo in S. cerevisiae. We determine that Pol ? depletion leads to a dependence on checkpoint signaling and recombination-mediated repair for cellular viability. By analyzing nascent lagging-strand products, we observe both a genome-wide change in the establishment and progression of replication forks and a global defect in Pol ?-mediated Okazaki fragment processing. Additionally, we detect significant lagging-strand synthesis by the leading-strand polymerase (Pol ?) in late regions of the genome when Pol ? is depleted. Overall design: Pol3 levels were altered by treatment with IAA in a strain containing an auxin-inducible degron on Pol3 and the resulting Okazaki fragments were sequenced from asynchronous and synchronous cultures. Addtionally, changes in ribonucleotide incorporation by ribonucleotide-hyperincorporating strains was assesed by HydEN-seq in order to determine the changes in polymerase usage during Pol3 depletion.